Project Significance: The faithful segregation of genetic material during cell division is critical for sustaining life. This becomes apparent if one considers that a 70-year old adult has used on average 1000 trillion (1020) cells, which were generated by an equal number of successful cell divisions. In contrast, chromosome segregation and cell division errors can lead to aneuploidy, yielding non- viable cells, genetic disorders or cancer. Cell division is orchestrated by the mitotic spindle, which is composed of a plethora of microtubules (MT) and MT associated proteins (MAP). First, the mitotic spindle captures and aligns chromosomes at the spindle equator. This is achieved via a bundle of MTs, which form the kinetochore-fiber (K-fiber) and connect the kinetochore (KT) of each sister chromatid with opposite cell poles. Next, sister chromatids are split and K-fibers pull sister chromatids to opposite cell poles. Despite 130 years of research on mitosis, it is not clear how the mitotic spindle is assembled and how it obtains its characteristic bipolar shape that is critical for accurate chromosome segregation. Most importantly, it is not understood how KTs are captured in the vast volume of a cell and aligned at its center. It also remains unknown how a threshold force is reached and transmitted via K-fibers to split and segregate sister chromatids and how errors occur. Answers to these questions are needed to explain how cells divide and thus procreate life, and may provide new treatments for diseases that lie at the heart of cell proliferation, such as cancer and cell regeneration. Here, I devise a holistic approach to overcome these experimental limitations and advance our understanding of chromosome segregation and its errors to a biochemical level. I will achieve this by breaking down the problem into the individual MT nucleation pathways that are required for spindle assembly and characterize these pathways at the single molecule level in vitro. This leaves us in the unprecedented position to start combining MT nucleation pathways. Thereby, we will build the mitotic spindle in vitro to not only to determine the molecular organization of the mitotic spindle, but also to elucidate the mechanisms by which K-fibers capture and segregate chromosomes and the causes of segregation errors. Finally, my research will explain how hundreds of proteins can self-assemble on the nm scale into a complex molecular machine 1000-fold larger than its constituents, a challenge for the biochemistry of the 21st century.